Gut microbiome associated with low anterior resection syndrome after rectal cancer surgery

This study aimed to assess the likely association of gut microbiome with low anterior resection syndrome (LARS) symptoms. Postoperative stool samples from patients with minor or major LARS after sphincter-preserving surgery (SPS) for rectal cancer were collected and analyzed using 16S ribosomal RNA sequencing method. The symptom patterns of LARS were classified into two groups (PC1LARS, PC2LARS) using principal component analysis. The dichotomized sum of questionnaire items (sub1LARS, sub2LARS) was used to group patients according to the main symptoms. According to microbial diversity, enterotype, and taxa, PC1LARS and sub1LARS were associated with frequency-dominant LARS symptoms and patients, while PC2LARS and sub2LARS were grouped as incontinence-dominant LARS symptoms and patients. Butyricicoccus levels decreased while overall LARS scores increased. The α-diversity richness index Chao1 showed a significantly negative correlation in sub1LARS and a positive correlation in sub2LARS. In sub1LARS, the severe group showed a lower Prevotellaceae enterotype and higher Bacteroidaceae enterotype than the mild group. Subdoligranulum and Flavonifractor showed a negative and a positive correlation with PC1LARS, respectively, while showing a negative relationship with PC2LARS. Lactobacillus and Bifidobacterium were negatively correlated to PC1LARS. Frequency-dominant LARS had decreased diversity of gut microbiome and showed lower levels of lactic acid-producing bacteria.

Only a few specific treatments with long-term effects have been reported 11 . To modulate postoperative bowel function, modifiable factors affecting bowel movements need to be investigated, and one of the modifiable factors associated with bowel function is the gut microbiome [14][15][16] . The gut microbiome affects colon motility and can be modified by diet, probiotics, and medications such as antibiotics [17][18][19][20][21] . Several studies have reported the likely association between gastrointestinal (GI) diseases and the gut microbiome in patients with irritable bowel syndrome [22][23][24] , inflammatory bowel disease 25 , and antibiotic-related Clostridium difficile colitis 26,27 . However, current research on the gut microbiome of LARS patients is scarce. Still, a previous report demonstrated that administering probiotics to LARS patients failed to improve LARS symptoms, but it positively modified serum immune markers 16 . Moreover, the association between the gut microbiome and LARS symptoms has not been studied. Understanding the gut microbiome of patients with LARS might provide a clue to the potential treatment of LARS patients with probiotics and/or prebiotics. Therefore, this study aimed to identify the gut microbiome of patients with LARS according to the main symptom groups and to compare the functional taxonomic differences between the groups.

Methods
Ethical approval of the study and informed consent. This was a retrospective cohort study based on the CRC cohort at Seoul National University Hospital. This cohort included patients who underwent surgery for CRC since 2014. The study design was approved by the Institutional Review Board (IRB) of the Seoul National University Hospital (IRB no. 1408-127-607). We followed the principles outlined in the Declaration of Helsinki. Informed consents were obtained from the study participants as the IRB approved.
Fecal sample collection from LARS patients. In our cohort for CRC patients, fecal samples were collected prospectively from patients between September 1, 2017, and May 31, 2019, after an average of 13.3 and 2.2 months following LAR and ileostomy repair, respectively. The stool samples were frozen and stored at − 20 °C. The fecal samples included in this study were collected from patients who underwent SPS for CRC from September 2013 to March 2019 and had minor or major LARS postoperatively (LARS score > 20). We included patients who experienced postoperative complications, and those with diverting stomas were excluded from the analysis. Surgeries were performed by surgeons in a single tertiary center where more than 500 colorectal cancer surgeries per year are performed in a standardized manner.
16S bacterial rRNA microbiota analysis. Whole DNA in fecal samples was extracted using the Mag-Bind Universal Pathogen 96 Kit (Omega Bio-Tek, Norcross, GA, USA) with a Hamilton Microlab STAR liquid handler (Hamilton Laboratory Solutions, Manitowoc, WI, USA) after bead-beating the samples with the Tissue-Lyser (Qiagen, Hilden, Germany), followed by amplicon PCR targeting the V3 to V4 region of the 16S bacterial rRNA gene using 341F and 805R primers (341F-CCT ACG GGNGGC WGC AG, 805R-GAC TAC HVGGG TAT CTA ATC C). After DNA library preparation, indexing and quality checks were performed using the Nextera XT index kit (Illumina, San Diego, CA, USA) and Qubit4.0 (Thermofisher, Wilmington, DE, USA), and 300× two paired-end sequencing was performed using the MiSeq system (Illumina, San Diego, CA, USA). The average read depth of the raw FASTQ files was over 50,000 counts. The denoised amplicon sequence variant (ASV) features acquired after DADA2 in QIIME2 2020.2 (qiime dada2) were classified at the taxonomic level by a pre-trained classifier with Silva138 28-31 . The sequenced raw data have been submitted in NCBI Sequence Read Archive with BioProject accession number PRJNA882613.
Definitions regarding LARS. The LARS questionnaire developed by Emmertsen et al. was used to assess postoperative bowel dysfunction following SPS 32 . Questionnaires were administered after an average of 13.3and 2.2-months following LAR and ileostomy repair, respectively. The questionnaire contained five items: flatus incontinence, liquid stool incontinence, frequency, clustering, and urgency. Each item comprised three severityweighted options. The LARS score was categorized into three groups: no LARS (0-20 points), minor LARS (21-29 points), or major LARS (30-42 points).
Grouping LARS using principal component analysis (PCA) and sum of scores of questionnaire items. Patterns with minor and major LARS were grouped using an approach with PCA performed on the five LARS questionnaire items. After extracting the principal components (PCs), varimax rotation was used to create a structure with independent variables and high potential for interpretability. The minimum eigenvalues of 1.0, screen plot, and interpretability of the factors were used to determine the variables that should be retained in the LARS patterns. As we did in previous studies, the symptoms of patients with LARS were clustered into two groups: incontinence-dominant and frequency-dominant 33 .
In addition to this PCA-based score, another subgroup scoring and classification were introduced to generalize the two dominant symptoms, as the PCA score is unique to this study's dataset.
Statistical and clustering analysis with microbiota data. The ASV feature table and taxonomic classification outputs were further processed using the phyloseq package in R to perform the diversity and taxonomic profiling analyses 34 . The total count normalization was performed with total sum scaling (known as relative abundance) without any ASV filtering step, except alpha-diversity richness inference, which uses singleton and doubleton count values. MaAsLin2 was used for taxonomic analysis at the genus level with continuous and categorical LARS-associated variables in this study after filtering taxa containing many zeros (zero in more than 90% of samples) 35

LARS-associated symptom subgroups.
Among 109 patients, 91 were classified into major LARS and 18 into minor LARS. The baseline characteristics of patients were shown in Supplementary Table S1. The mean age was 62.2 years, and 101 (92.7%) of patients were males. The mean tumor location from the anal verge was 7 cm, and 58.7% of patients had diverting ileostomy followed by ileostomy reversal. Preoperative or postoperative radiotherapy was administered in 55 (50.5%) patients, and 3 (2.8%) patients experienced anastomotic leakage. The questionnaire items for frequency, clustering, and urgency were grouped and contributed to the first PC variable (PC1LARS) in the PCA. Others, including liquid stool incontinence and flatus incontinence, accounted for a greater proportion of the second PC variable (PC2LARS) (Fig. 1).
In addition, the sum of scores for items regarding frequency, clustering, and urgency was referred to as sub-1LARS, while the sum of scores for incontinence for flatus and liquid stools was referred to as sub2LARS. Therefore, similar to the previous research 33 , the PC1LARS and sub1LARS corresponded to the frequency-dominant LARS, and the PC2LARS and sub2LARS corresponded to the incontinence-dominant LARS. Table 1 summarizes the description of these LARS-associated variables and the range of each variable. For the totalLARS, pre-defined indicators of LARS questionnaires were applied. To further interpret the new symptombased LARS subgroups, we introduced grouping criteria for each LARS-associated variable. In the case of subgrouping items in the LARS questionnaires, binary group variables derived from the new continuous variables were devised. The criteria applied to each level are shown in the "levels" column of this table. Supplementary Table S2 summarizes the distribution of the LARS-associated variables.
The cutoff value of LARS-associated binary variables was determined by considering the distribution of patients. In the sub1LARS, the "severe" classification was determined when bowel frequency was more than four times a day together with clustering symptom and urgency at least once a week. In the sub2LARS, "severe" refers to symptoms of incontinence for both flatus and liquid stools that occur at least once per week (Supplementary Table S3). The two group factors were significantly independent of each other (chi-square = 0.410, P = 0.522).
Microbial diversity. Microbial alpha diversity was measured using different methods for richness and diversity in order to compare the LARS groups (Table 2). No significant correlation was noted between the total-LARS score and all alpha-diversity indices, whereas a significant correlation was noted between the sub1LARS score or sub2LARS and alpha diversity richness. Briefly, the microbial richness index, including Observed feature number and Chao1, was monitored, revealing a significantly negative correlation in sub1LARS Severity of LARS according to enterotypes of the gut microbiome. The gut microbiome was clustered into three enterotypes, to which the main bacterial taxa contributing were Prevotellaceae, Ruminococcaceae, and Bacteroidaceae at the Family level (Fig. 3a). A different composition was identified in the sub-1LARS_group compared with sub2LARS_group and totalLARS_group (Fig. 3b). In the severe sub1LARS_group, the proportion with Bacteroidaceae enterotype was higher, and those with Prevotellaceae enterotype tended to be lower than those in the mild sub1LARS_group (P = 0.692). In contrast, sub2LARS_group and totalLARS_group showed higher Prevotellaceae enterotype in the severe group than in the mild group (P = 0.625 and P = 0.311, respectively).
Differentially abundant taxa between LARS groups. Twenty genera among a total of 123 genera showed a significant relationship (the absolute value of Spearman's rank coefficient was above 0.2, nominal P < 0.05) to at least one variable among the LARS-associated continuous variables (Fig. 4).

Discussion
This study is the first to reveal the gut microbiome of LARS patients and to identify taxonomic differences in relation to dominant symptoms. As reported previously 33 , the main symptoms of LARS were clustered into two groups: frequency-dominant and incontinence-dominant. In the LARS questionnaire, frequency-dominant symptoms were expressed through questions regarding clustering, urgency, and frequent bowel movements, www.nature.com/scientificreports/ whereas incontinence-dominant symptoms were expressed through fecal and flatus incontinence. The questions were classified into two groups, and some taxa of the gut microbiome were differentially expressed according to the two dominant LARS symptom groups. The Prevotellaceae enterotype and lactic acid producers, including Lactobacillus and Bifidobacterium, were at lower levels in the sub1LARS_group with severe frequency, clustering, and urgency. Severe sub1LARS_group had lower alpha diversity compared to mild sub1LARS_group. High richness and diversity of the gut microbiome are considered characteristics of a healthy gut microbial ecosystem, which reflects    Sub1LARS_group had a distinctive taxonomic composition. The frequent bowel movements and evacuatory dysfunctions in LARS patients may be associated with the gut microbiome. Therefore, modification of the microbiome could adjust these dysfunctions. The autonomic and enteric nervous systems control the bowel motility of the distal colon and rectum, while the autonomic nerve fibers to the distal colon, which becomes the neorectum after LAR, are transected 11 . Due to the destruction of an inhibitory alpha-sympathetic pathway, extrinsic denervation results in increased motility, which may account for the frequent bowel movements observed in LARS patients 41 . The enteric nervous system subsequently controls the neorectum, and serotonin (5-HT) receptors allow intrinsic primary afferent neurons to sense and regulate the enteric nervous system. 5-HT receptor activity can be modulated by the serotonin receptor antagonist ramosetron, commonly prescribed to LARS patients 42 ; furthermore, LARS may also be regulated by the gut microbiome due to its previous reported ability to modify the levels of 5-HT in the colon 19,21 .
Some studies have reported symptom improvement in LARS patients using probiotics. However, they had several limitations. A randomized controlled trial (RCT) administered probiotics during 4 weeks to LARS patients in order to improve bowel function following an ileostomy closure but failed to demonstrate any improvement in the postoperative GI QOL index of patients 15 . Another RCT using a single-strain probiotic, Lactobacillus plantarum DJLP243, also failed to demonstrate significant effects on bowel dysfunction following ileostomy reversal 14 . These studies were conducted during the perioperative period of ileostomy reversal when patients experienced the most severe intestinal dysfunction following stoma restoration, and gut microbiome changes were not analyzed. Our study included only patients who completed acute cancer care, and their gut microbiome was comprehensively analyzed to understand the relationships between the gut microbiome and their symptoms.
Although the aforementioned RCTs did not demonstrate significant improvements in bowel dysfunction after ileostomy reversal, these studies were initiated based on the hypothesis that the gut microbiome is related to bowel motility and GI functional disorders, which was supported by previous studies [18][19][20][21] . 5-HT (a key regulator of GI motility) levels in the colon epithelium and lumen are influenced by short-chain fatty acids produced by gut microbiota via tryptophan hydroxylase 1 in enterochromaffin cells in the lining of the GI tract 18,21 . Neuronal reprogramming is an additional hypothesis explaining the relationship between the gut microbiome and GI function 19,20 . Therefore, unveiling the gut microbiome of LARS patients will help develop ways to improve LARS.
This study was the first to comprehensively analyze the gut microbiome of patients at the diversity, enterotype, and species levels and to compare microbiome taxa according to predominant symptom-based LARS groups. Patients with incontinence-dominant LARS (sub2LARS_group) may have an injury to the internal sphincter; therefore, the improvement of the symptoms by means of the modulation of gut mobility with probiotics is unlikely. However, the frequency-dominant LARS (sub1LARS_group) exhibited decreased alpha diversity and low number of lactic acid-producing bacteria, including Lactobacillus and Bifidobacterium, which are commonly www.nature.com/scientificreports/ produced as probiotics, suggesting that probiotic induced microbiome modulation may benefit the sub1LARS_ group to alleviate LARS symptoms. This microbiome analysis study may serve as the basis for future microbiome modulation studies specifically targeting the sub1LARS_group. This study has some limitations. First, postoperative dietary changes might influence the gut microbiome composition because patients attempt to adapt to LARS-associated symptoms. Based on past experiences, individuals with severe frequency-related symptoms will attempt to consume foods that alleviate symptoms. Moreover, such food differences among groups might influence the gut microbiome, although no dietary information was collected in this study. Second, whether the microbiome in the gut was the cause of the LARS symptoms was unclear.
As this was an observational study and not an interventional one, we were unable to explain the causality of the results. Third, we included only patients with LARS symptoms. The patients in this study were almost male with low-lying rectal cancer who received radiotherapy pre or postoperatively. Therefore, the patients might have deviated characteristics from the standard group of rectal cancer patients, which could bias the results. Because we aimed to elucidate the relationship between LARS symptoms and microbiome, we only included patients with LARS symptoms. To know the sole effect of microbiome on LARS considering the other interference of host and treatment related factors, we need further research.
In conclusion, LARS was classified into frequency-dominant and incontinence-dominant LARS groups. Patients with severe frequency-dominant LARS had decreased microbial diversity and exhibited a higher Bacteroidaceae enterotype, a lower Prevotellaceae enterotype, and fewer lactic acid-producing microorganisms, including Bifidobacterium and Lactobacillus than the non-severe frequency-dominant LARS patients, which was not observed in the incontinence-dominant LARS group. Future studies are needed to evaluate the effects of Lactobacillus and/or Bifidobacterium in patients with frequency-dominant LARS to monitor the alleviation of LARS symptoms.